Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin... Show moreSkeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short -chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mito-chondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate -induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism. Show less
Ongay, S.; Langelaar-Makkinje, M.; Stoop, M.P.; Liu, N.; Overkleeft, H.S.; Luider, T.M.; ... ; Bischoff, R. 2018
Formaldehyde fixation is widely used for long-term storage of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering mass spectrometry ... Show moreFormaldehyde fixation is widely used for long-term storage of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering mass spectrometry (MS)-proteomic analyses. In the present work we evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation as well as efficient protein extraction for MS-proteomics under mild conditions. We investigated disuccinimidyl tartrate (DST) and dithiobis[succinimidylpropionate] (DSP) as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups leading to amide bond formation and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP). Our results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to formaldehyde.Cleavage of DSP improves protein recovery from fixated tissue by a factor of 18 and increases the number of identified proteins by approximately 20% under mild extraction conditions compared to formaldehyde-fixated paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, while effective, resulted in side reactions that prevented effective protein extraction and interfered with protein identification. Show less
